A hypoxia-responsive ionizable lipid

Abstract

Provided herein is an ionizable lipid and a lipid nanoparticle composition including the ionizable lipid. The ionizable lipid and lipid nanoparticle composition are hypoxia responsive. Provided herein are also methods of delivering an agent to hypoxic tissue and treating disorders associated with hypoxia using the ionizable lipid and lipid nanoparticle composition. Such methods include treatment of placental related disorders.

Description

A HYPOXIA-RESPONSIVE IONIZABLE LIPIDCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 676,983, filed on July 30, 2024, which is hereby incorporated in its entirety.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Federal government support under contract numbers 2301919, awarded by the Engineering Research Initiation. The Federal government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure relates to an ionizable lipid, and it’s use in a lipid nanoparticle formulation that increases RNA delivery to cells in a low oxygen environment.BACKGROUND OF THE INVENTION

[0004] Lipid nanoparticles (LNPs) have gained rapid attention as mRNA delivery vehicles for vaccination against SARS-CoV-2 and other diseases. mRNA is a potent therapeutic tool because it enables transient protein production; however, it is easily degraded by serum endonucleases and has limited cellular uptake. LNPs encapsulate nucleic acids, offering several benefits including increased mRNA stability, circulation time, cell uptake, and tissue specificity (N. Chaudhary, et al. Nat Rev Drug Discov, 20 (2021)). Altering the type and molar ratios of each component of the LNPs strongly influences tissue- and cellspecificity (K.J. Kauffman, et al. Nano Lett, 15 (2015)). LNPs capable of delivering agents, such as nucleic acids, for example RNA, to low oxygen environments, also known as hypoxic environments or hypoxia, would be particularly useful in treating low oxygen environment disorders, such as cancer, e.g. solid tumors, and placental -related disorders, for example preeclampsia.

[0005] Placental -related disorders, such as preeclampsia, are the leading cause of maternal mortality worldwide. Placental dysfunction results in anti-angiogenic and inflammatory factors released into the maternal circulation, which can lead to systemic vasoconstriction, e.g. maternal hypertension, and fetal growth restriction, e.g. impaired blood supply to the fetus which can compromise fetal growth. Currently, the only effectivetreatment option includes preterm delivery of the placenta and fetus, which has many detrimental effects to the developing fetus. At early stages of pregnancy, e.g. <28 weeks, delivery of the placenta and fetus has low fetal survival rates due to underdevelopment of the lungs and other major organs. Extending the duration of pregnancy to a safer time for delivery, e.g. ideally >34 weeks, may offer a means to treat placental disorders, improving outcomes for both the fetus and pregnant subject. For example, preeclampsia is one of the leading causes of maternal death during pregnancy, causing severe hypertension and proteinurea. Preeclampsia can lead to other complications, such as kidney failure and heart disease following pregnancy.

[0006] Labetalol, an anti-hypertensive drug, is approved for use during pregnancy; however, dosing is limited due to potential fetal growth restriction and it only treats a symptom of preeclampsia, i.e. hypertension, not the placenta itself. Other beta blockers and calcium channel blockers have increased risk of fetal growth restriction and perinatal mortality. Thus, there remains a significant need for safe and effective treatment options for placental -related disorders.SUMMARY OF THE INVENTION

[0007] This disclosure is based, at least in part, on at least the following surprising findings:(1) a novel ionizable lipid that is hypoxia responsive.(2) a lipid nanoparticle (LNP) composition comprising four lipid components: the novel ionizable lipid, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), cholesterol, and (l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)) (DMPE-PEG).(3) the LNP composition preferentially targets low oxygen environments, i.e. hypoxic or hypoxia, over normal oxygen environments, i.e. normoxia, and can deliver agents, such as mRNA, siRNA, microRNA, short hairpin RNA (shRNA), DNA, proteins, peptides, etc. Thus, this disclosure provides a novel method of delivery of agents to low oxygen environments and methods of treating disorders associated with hypoxia.

[0008] The present invention provides an ionizable lipid comprising a head group, a polyamine linker, and a hydrocarbon tail. The head group comprises a nitroimidazole moiety,the polyamine linker contains tertiary amine groups, and the tail includes a hydrocarbon chain of 8, 10, 12, 14, 16, or 18 carbon atoms. These ionizable lipids are responsive to hypoxic conditions, thereby enabling increased delivery to hypoxic environments.

[0009] In a preferred embodiment, the ionizable lipid comprises 2-nitroimidazole as the head group, 1,3 -diaminopropane as the polyamine linker, and 1,2-epoxy dodecane as the hydrocarbon tail.

[0010] The invention further provides a lipid nanoparticle (LNP) composition including the ionizable lipid described herein, l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol, and DMPE-PEG (l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)). The components are present in specific weight ratios, for example to enhance stability and delivery performance. In embodiments, the LNP composition includes about 25% to about 45% of the ionizable lipid, about 10% to about 22% of DOPE, about 30% to about 60% cholesterol, and about 0.5% to about 3.5% DMPE-PEG. In a preferred embodiment, the LNP composition includes about 35% ionizable lipid, about 10% DOPE, about 53.5% cholesterol, and about 1.5% DMPE-PEG.

[0011] The LNP composition may further include at least one agent, such as, without limitation, a nucleic acid, e.g. mRNA, siRNA, microRNA, shRNA, DNA, etc., or peptide, protein, etc. The LNP composition may incorporate or encapsulate the agent. In some embodiments, mRNA is the preferred agent. The ratio of total agent to total LNP composition is specific to the agent. In an embodiment the ratio of total agent, e.g. mRNA, to total LNP is about 1 :5 (w / w) to about 1 : 15 (w / w).

[0012] Also provided herein is a method for treating a disorder associated with hypoxia in a subject by administering the LNP composition and a therapeutic agent to the subject in need thereof. In one example embodiment, the disorder is a placental -related disorder, such as preeclampsia.

[0013] The invention also provides a method for delivering at least one agent to hypoxic tissue in a subject by administering the LNP composition incorporating at least one agent described herein to the subject in need thereof, resulting in the agent(s) being delivered preferentially to the hypoxic tissue. In certain embodiments, the hypoxic tissue is the placenta.

[0014] Other embodiments will become apparent from a review of the ensuing detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1 shows sFlt-1 secretion (left) and P1GF secretion (right) in normoxia and hypoxia in HTR8 and BeWo cells.

[0016] Fig. 2 depicts the chemical structures of 2-NI-K3-E12 (A) and 2-MI-K3-E12 (B).

[0017] Fig. 3 shows a flash chromatography spectrum of raw product from reaction mixture. Separation yielded 6 distinct peaks.

[0018] Fig. 4 shows 'H NMR spectra of all peaks from flash chromatography stacked on top of each other.

[0019] Fig. 5 shows a1H NMR spectra of isolated peak 2 from flash chromatography spectra, identified as 2-NI-K3-E12. Annotations show each part of the lipid.

[0020] Fig. 6 shows normalized fluorescense plotted against pH for TNS assay data on NI-, MI-, and C12-LNPs.

[0021] Fig. 7 shows normalized luminescence (left) in normoxia and hypoxia, and cell viability (right) following LNP treatment in (A) HTR8 cells, representative of first trimester and (B) BeWo cells, representative of third trimester.DETAILED DESCRIPTION

[0022] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0024] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 to 8 is stated, 2, 3, 4, 5, 6, and 7 are also intended to be explicitly disclosed, as well as the range of values greater than or equal to 1 and the range of values less than or equal to 8.

[0025] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0026] As used herein, the terms “patient” or “subject” are used interchangeably and refer to an animal, including a mammal, preferably a human.

[0027] As used herein, the terms “treat,” “treatment,” and “treating” in the context of the administration of a therapy to a subject refer to the reduction or inhibition of the progression and / or duration of a disease or condition and / or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies.

[0028] As used herein, the terms composition(s) and formulation(s) are used interchangeably.

[0029] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0030] The present disclosure relates to a novel ionizable lipid and its use in lipid nanoparticle (LNP) formulations to provide increased delivery of agents, such as without limitation mRNA, siRNA, microRNA, short hairpin RNA (shRNA), DNA, proteins, etc. to low oxygen environments. The disclosed lipid, compositions, and methods are useful for development of and / or as therapeutics to treat disorders associated with low oxygen environments, such as preeclampsia.

[0031] In embodiments, the disclosure relates to an ionizable lipid comprised of a head, linker, and tail groups, which provides a versatile platform for creating a range of compounds. In one embodiment the head includes a nitroimidazole. In a particular example embodiment, the hypoxia-responsive head group is 2-nitroimidazole (2-NI). 2-NI has previously been investigated as an imaging agent for tissue hypoxia, antibiotic drug for infections, and radiosensitizer for cancer treatments. A few research groups have conjugated 2-NI to polymers or lipids to create hypoxia-responsive drug carriers. However, often 2-NI is conjugated to the tails of the lipid. Alternatively, this disclosure relates to utilizing 2-NI as a head group for an ionizable lipid, while the linker and tail can be interchanged with various linker and tail structures to customize the lipid’s design. The head group responds to hypoxia by undergoing chemical reduction when there is low oxygen. The modular nature of the ionizable lipid structure allows for customization of the linker and tail to optimize the lipid for specific applications, while maintaining the head structure for its hypoxia-responsive properties. In embodiments, the linker is a polyamine linker, for example with secondary or tertiary amines. In a preferred embodiment, the polyamine linker contains tertiary amines,such as those formed when synthesized with 1,3 -diaminopropane. The tail group may be a saturated or unsaturated hydrocarbon chain. In an example embodiment, the hydrocarbon tail length may include 8 to 18 carbons in increments of two, i.e, 8, 10, 12, 14, 16, or 18. In an embodiment, the tail group is a saturated 12 carbon tail, for example, 1,2-epoxy dodecane.

[0032] In one embodiment, the lipid comprises 2-nitroimidazole as the head group, 1,3-diaminopropane as the linker, and 1,2-epoxydodecane as the tail. In this particular embodiment, the lipid, sometimes called 2-NI-K3-E12, has the following structure:

[0033] In embodiments, the linker and tail groups in the ionizable lipid structure could be changed or modified based on desired properties. For example, the linker could be modified to include more amine groups to increase the charge of the lipid after ionization. Changing the charge of the lipid could improve RNA encapsulation and / or improve RNA delivery when the lipid is formulated into a LNP composition. The hydrocarbon tail length and saturation level could also be modified to change LNP composition delivery properties.

[0034] In other embodiments, the disclosure relates to a novel ionizable lipid nanoparticle (LNP) composition comprising four lipid components: the disclosed novel ionizable lipid described herein, for example 2-NI-K3-E12, l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol, and (l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)) (DMPE- PEG). The ionizable lipid may be about 25 to about 45% of the total composition. The 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) may be about 10 to about 25% of the total composition. The cholesterol may be about 30 to about 60% of the total composition. The (l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (ammonium salt)) (DMPE-PEG) may be about 0.5 to about 3.5% of the total composition. In one embodiment the LNP composition comprises about 35% of the disclosed ionizable lipid, about 10% l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), about 53.5% cholesterol, and about 1.5% (l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)) (DMPE-PEG). The LNP composition can be modified to optimize delivery to tissues and cells, specifically the four lipid components can be mixed together at various molar ratios.

[0035] Hypoxia-responsive LNP compositions are superior to other LNP compositions because they will increase LNP composition delivery to hypoxic sites in the body. Increased LNP composition delivery means there will be increased delivery of agents, e.g. RNA, to cells, which is expected to increase the therapeutic effect of the agent being delivered. Most tissue-targeting LNP compositions utilize an antibody fragment or peptide conjugated to the PEG on the surface of LNP compositions to target the tissue site. However, the targeting capacity of these LNP compositions is limited by protein corona formation on the LNP composition surface and dissociation of PEG molecules following injection into the body. The disclosed LNP compositions passively target hypoxia utilizing the hypoxia- responsive ionizable lipid without the use of a conjugated ligand. An additional benefit of the disclosed lipid includes incorporation of 2-NI in the head group allowing for the ionizable lipid to maintain an inverted hexagonal phase structure in the LNP formulation when exposed to acidic conditions. This lipid packing structure allows LNP compositions to have increased endosomal escape inside cells. In embodiments, the LNP composition may be conjugated with a ligand to the LNP surface for more specific targeting.

[0036] In additional embodiments, the disclosure relates to methods for delivering agents, such as messenger RNA (mRNA) or small interfering RNA (siRNA), microRNA, short hairpin RNA (shRNA), DNA, proteins, peptides, etc. to low oxygen environments. At least one agent may be added to the LNP composition, for example by dilution, at a preferred w / w ratio for the specific agent(s). In some examples, the agent may be encapsulated by the LNP. In embodiments, the composition includes the at least one agent, for example RNA, e.g. mRNA, at a total RNA : total LNP ratio of about 1 :5 to about 1 : 15 (w / w), such as about 1 :9 to about 1 : 11 (w / w) or about 1 : 10 (w / w). Other embodiments relate to treating disorders associated with hypoxia, such as cancer, e.g. solid tumors, and placental -related disorders, for example preeclampsia, by administering an LNP composition encapsulating or incorporating at least one therapeutic agent to a subject in need thereof.

[0037] In other embodiments methods of treating placental -related disorders, for example without limitation placental insufficiency, fetal growth restriction, and preeclampsia, by delivering at least one therapeutic agent, such as mRNA or siRNA, to placental cells, e.g. trophoblasts, and / or the placenta through administration of the LNP formulations to a subject in need thereof are disclosed. Example agents include, without limitation, placental growth factor (P1GF) mRNA, vascular endothelial growth factor (VEGF) mRNA, soluble fms-liketyrosine kinase- 1 (sFlt-1) siRNA, and soluble endoglin (sEng) siRNA. In one embodiment, the disclosure relates to treating preeclampsia. For example, an LNP formulation including placental growth factor (P1GF) mRNA and / or soluble fms-like tyrosine kinase-1 (sFlt-1) siRNA is administered to treat preeclampsia in a subject in need thereof. The LNP formulations may be used to treat and / or deliver a therapeutic agent, such as mRNA, to a pregnant subject in need thereof after 20 weeks and prior to 37 weeks of pregnancy.

[0038] While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.EXAMPLES

[0039] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.Example:

[0040] Previously, a design of experiments (DOE) approach was used to identify an LNP that yielded high mRNA delivery to the placenta to treat pregnancy-related diseases, such as preeclampsia (PE) (R.E. Young et al. Bioact Mater, 34 (2024)). During PE, dysregulated trophoblast invasion and spiral artery remodeling causes poor placental development and insufficient perfusion, leading to oxidative stress (G.J. Burton et al. Reproduction, 161 (2021)). Oxidative stress in the placenta prevents proper angiogenesis, leading to hypertension characteristic of PE (N. Soleymanlou et al. J Clin Endocrinol Metab, 90 (2005)). Here, how high oxidative stress throughout pregnancy impacts LNP-mediated drug delivery was studied. This understanding could yield next-generation platforms for improved delivery to the diseased placenta. First, delivery of mRNA was examined with our LNP library in multiple trophoblast cell lines. Then, how hypoxia (1% 02) influences trophoblast behavior to establish an in vitro model representative of oxidative stress in PE was investigated. Hypoxic environments influenced trimester-specific trophoblast behavior (Figure 1). Hypoxia increased growth, decreased syncytialization, and decreased LNP uptake in first and third trimester trophoblasts. To improve delivery to the diseased placenta, nextgeneration LNPs were developed that are chemically designed to respond to hypoxia. A new approach to target LNPs to the diseased placenta and mechanistic insights into the role ofoxygen tension in disease progression was provided. Further, the hypoxia-responsive lipids provide a new LNP targeting strategy for a myriad of diseases implicated by low oxygen.

[0041] Synthesis of 2-NI-K3-E12: 2-nitroimidazole (2-NI, 2.50 g, 22 mM, 1 eq) was added to methyl acrylate (2.284 g, 26.5 mM, 1.2 eq) in 10 mL of methanol in a reaction flask. Mixture was covered and stirred for 48 hours at 80 C. Solvent was evaporated using vacuum. 1,3-diaminopropane (K3, 3.278 g 44 mM, 2 eq) was added to the reaction flask in 10 mL of methanol. The mixture was covered and stirred for 5 days at room temperature. Excess 1,3- diaminopropane was removed by washing the mixture three times with 5% methanol in diethyl ether. The organic phase was removed by vacuum evaporation. 1,2-epoxy dodecane (E12, 16.301 g, 88 mM, 4 eq) was added neat to the reaction flask. Mixture was covered and stirred overnight at 90 C covered in foil. The resultant reaction mixture was collected into an amber bottle and stored at 4 C. The resultant product is the hypoxia-responsive lipid, 2-NI- K3-E12 (Figure 2A). The ionizable lipid consists of a hypoxia-responsive head group, amine linker, and hydrocarbon tail. A chemically similar, non-responsive, ionizable lipid was fabricated the same way, except 2-methylimidzaole (2-MI, 2.50 g 30 mM, 1 eq) was used instead of 2-NI. The resultant ionizable lipid is 2-MI-K3-E12 (Figure 2B).

[0042] Purification of 2-NI-K3-E12: 2-NI-K3-E12 was purified by flash chromatography using an 80 g silica cartridge and a solvent gradient from 100% di chloromethane (DCM) to 100% ULTRA with auto holds on solvent gradient enabled. ULTRA is a polar solvent comprised of a mixture DCM : methanol : ammonium hydroxide (30 in H2O) at 75:22:3 volumetric ratio. All peaks (Figure 3) were collected separately, and excess solvent was evaporated.

[0043] Characterization of 2-NI-K3-E12:JH NMR for each peak from the flash chromatography spectra were recorded and referenced to methanol-d4 at 3.35 ppm and 4.80 ppm using a Bruker Avance III system (Figure 4). Upon analysis, peak 2 from the flash chromatography spectra was identified as 2-NI-K3-E12 (Figure 5).

[0044] Formulation of LNPs: Each LNP is formulated via mixing with micropipettes, combining one volume of lipid / ethanol mixture to three volumes of mRNA in citrate buffer with a pH of 3 (1 :3 ethanol : citrate volume ratio). The lipid mixture for each LNP contains a molar ratio of ionizable lipid : phospholipid : cholesterol : PEG of 35: 10:53.5: 1.5. LNPs were made with 2-NI-K3-E12 (NI-LNP), 2-MLK3-E12 (MI-LNP), or C12-200 (C12-LNP). C12-200 was synthesized by our laboratory and purified by flash chromatography, as described above. The chemical structure of C 12-200 was designed by a different laboratory. mRNA was diluted in citrate buffer to an mRNA : ionizable lipid ratio of1 : 10 (w / w) for each of the LNP formulations. Codon optimized mRNA was prepared by in vitro transcription through a collaboration with the Engineered mRNA and Targeted Nanomedicine core facility at the University of Pennsylvania. Firefly luciferase was cosynthesized with 1 -methylpseudouridine modifications, and co-transcriptionally capped using the CleanCap system (TriLink) and purified using cellulose based chromatography. Following mixing of the lipid / ethanol and mRNA / citrate phases, the LNPs were dialyzed against PBS (pH 7.4) for 2 hours, sterile filtered using a 0.2 pm filters, and stored at 4°C.

[0045] Characterization of LNPs: NI-, ML, and C12-LNPs were characterized in the same way and values reported in Table 1.Table 1. Diameter mRNA Encapsu ation Zeta PotentiaLNP . . PDI / o / .rpKa . ...(nm) (%)r(mV)Nl 154.0 ± 1.5 0.1995 56.3 ± 0.88 6.597 5.73 ± 0.70Ml 158.8 ± 2.5 0.1961 53.7 ± 0.032 6.444 10.2 ± 1.56C12 129.6 ± 0.85 0.1590 58.2 ± 0.36 6.880 15.6 ± 3.13

[0046] Each LNP was diluted 1 : 100 in deionized water in folded capillary cuvettes. Dynamic light scattering (DLS) measurements and zeta potential were recorded on a Malvern Zetasizer Nano ZS with an applied voltage of 150 volts. The encapsulation efficiency of each LNP formulation was calculated using QuantiFluor® RNA System. Briefly, LNPs were diluted 1 : 100 in IX TE buffer in two microcentrifuge tubes per LNP formulation. 1% v / v Triton X-100 was added to one of the tubes and both were heated to 37°C and shaken at 600 RPM for 5 mins, followed by cooling to room temperature for 10 mins. LNP samples and RNA standards were plated in triplicate in black 96-well plates and the fluorescent reagent was added per the manufacturer instructions. Fluorescent intensity was read on the plate reader (excitation, 492 nm; emission, 540 nm). Background signal was subtracted from each well and triplicate wells for each LNP were averaged. RNA content was quantified by comparing samples to the standard curve, and encapsulation efficiency (%) was calculated according to the equation EE=(B - A ) / Bx 100, where A is the RNA content in samples without Triton X-100 treatment (intact LNPs) and B is the RNA content in samples treated with Triton X-100 (lysed LNPs). The apparent pKa of LNPs was determined via TNS [6-(p- toluidinyl)naphthalene-2-sulfonic acid] assays. Briefly, a buffer solution of 150 mM sodiumchloride, 20 mM sodium phosphate, 20 mM ammonium acetate, and 25 mM ammonium citrate (VWR Chemicals BDH, Radnor, PA) was separated into 21 varied pH solutions, adjusted from pH 2 to 12 in increments of 0.5 pH. 2.5 pL of each LNP formulation was combined with 125 pL of each pH-adjusted solution in black 96-well plates in triplicate. TNS was added to each well for a final TNS concentration of 6 pM and the fluorescence intensity was read on a plate reader (Molecular Devices, San Jose, CA) (excitation, 322 nm; emission, 431 nm). Fluorescence intensity versus pH was plotted, and apparent pKa was calculated as the pH corresponding to 50% of its maximum value, representing 50% protonation (Figure 6).

[0047] In vitro transfection with LNPs: NI-, MI-, and C12-LNPs were formulated with luciferase mRNA as a reporter molecule and a luciferase assay was performed to measure transfection and mRNA translation in two trophoblast cell lines. The b30 clone of the BeWo choriocarcinoma cell line (termed “BeWo” herein) were cultured in F-12K Nutrient Mixture (Kaighn's Mod.) with L-glutamine supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. BeWo cells are derived from human choriocarcinoma and are representative of a third trimester placental cell line. HTR-8 / SVneo cells (termed “HTR8” herein) were cultured in RPMI 1640 with 2.05 mM L- glutamine supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. HTR8 cells are derived from transfecting cells from human chorionic villi explants with simian virus 40 large T antigen and are representative of a first trimester placental cell line. Cultures were grown in normoxia or hypoxia. Normoxia conditions were an incubator with temperature set at 37 C with 5% CO2 in room air (21% O2). Hypoxia conditions were created using a hypoxic cell culture chamber (StemCell Technologies) purged with low oxygen gas (1% O2, 5% CO2, and 94% N2) for 5 minutes. The cell culture chamber was then placed in a warm room with temperature set at 37 C. Cells were plated at 12,500 cells / cm2in 24-well plates with 1 mL of complete culture media in triplicate for each LNP formulation for each oxygen condition. After 4 hours of attachment in normoxia, cell culture media was replaced and the cells for hypoxia were placed in the hypoxic cell culture chamber, purged with low oxygen gas, and put in the warm room. After 48 hours of culture, cells were treated with each LNP at a 0.5 nM (250 ng / well) dose of mRNA. After 24 hours treatment with LNPs, cells were collected using 0.25% trypsin with 0.2 g / L EDTA to lift them from the plate surface and complete media to inactivate the trypsin after lifting. Cells were washed with PBS, counted, and diluted to 500,000 cells / mL in IX cell lysis buffer. After 10 minutes at room temperature, cell lysates were centrifuged at 10,000 x g for 10 minutes. 20 pL of lysates were plated into white 96-well plates. 100 pL of luciferase assay substrate (Promega) was added to each well and the luminescent signal was quantified using the plate reader (Figure 7).

[0048] In vitro viability following LNP treatment: BeWo and HTR8 cells were cultured in normoxia as described above. Cells were plated at 12,500 cells / cm2in 96-well plates with 200 pL of complete culture media in triplicate for each LNP formulation. Cells were treated with two doses of NI-, MI-, and C12-LNPs, 25 and 50 ng mRNA / well. To assess metabolic activity as an indicator of cell viability, after 24 hours treatment with LNPs, cells were assayed using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] reduction assay (CellTiter 96 AQueous One Solution, Promega). 20 pL of MTS reagent was added to each well. After incubation for 2 hours at 37 C, absorbance at 590 nm was measured on the plate reader. The average absorbance of wells containing no cells was subtracted as background from each well. The absorbance signal from each group was normalized to untreated cells and reported as the fold change in absorbance (Figure 6).

[0049] In conclusion, a DOE was utilized to examine a library of LNPs in first- and third trimester trophoblast cell lines, which resulted in a top formulation for each. LNPs with DOPE increased delivery compared to DSPC. When cultured in hypoxia, all cell lines had elevated HIF-la levels and increased cell growth. Further, third trimester cell lines, JAR and BeWo, had decreased syncytialization after culture in hypoxia, Lastly, a hypoxia-responsive ionizable lipid, 2-NI-K3-E12, was developed to increase delivery of LNPs in hypoxic environments, such as the preeclamptic placenta.

[0050] These new lipids increased delivery to cells cultured in hypoxia 2.2-fold compared to non-responsive LNPs. The ionizable lipid, combined with top LNP design parameters for each cell line, will be utilized to study and treat diseases implicated by low oxygen environments, or hypoxia, such as some cancers and placental related disorders, for example PE.

Claims

CLAIMSWhat is claimed is:

1. An ionizable lipid comprising a head group, a polyamine linker, and a tail, wherein the head group comprises a nitroimidazole, the polyamine linker comprises tertiary amine groups, and the tail comprises a hydrocarbon chain of 8, 10, 12, 14, 16, or 18 carbons.

2. The ionizable lipid of claim 1, wherein the head group is 2-nitroimidazole, the polyamine linker is 1,3-diaminopropane, and the tail is 1,2-epoxy dodecane.

3. The ionizable lipid of claim 1, wherein the lipid is hypoxia responsive.

4. A lipid nanoparticle composition comprising the ionizable lipid of claim 1, 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and (1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy (poly ethylene glycol)-2000] (ammonium salt)) (DMPE-PEG).

5. The composition of claim 4, wherein the ionizable lipid comprises 2-nitroimidazole as the head group, 1,3-diaminopropane as the polyamine linker, and 1,2-epoxy dodecane as the hydrocarbon tail.

6. The composition of claim 4, wherein the ionizable lipid is about 25% to about 45% of the total composition, DOPE is about 10% to about 22% of the total composition, cholesterol is about 30% to about 60% of the total composition, and DMPE-PEG is about 0.5% to about 3.5% of the total composition.

7. The composition of claim 6, wherein the ionizable lipid is about 35% of the total composition, DOPE is about 10% of the total composition, cholesterol is about 53.5% of the total composition, and DMPE-PEG is about 1.5% of the total composition.

8. The composition of claim 4, further comprising at least one agent.

9. The composition of claim 8, wherein the agent is selected from the group consisting of a mRNA, siRNA, microRNA, short hairpin RNA (shRNA), DNA, peptide, and protein.

10. The composition of claim 8, wherein at least one agent is mRNA.

11. The composition of claim 8, wherein the ratio of total agent to total lipid nanoparticle is about 1 :5 to about 1 : 15 (w / w).

12. A method for treating a subject having a disorder associated with hypoxia comprising administering the composition of claim 8 to a subject in need thereof.

13. The method of claim 12, wherein the ionizable lipid comprises 2-nitroimidazole as the head group, 1,3-diaminopropane as the polyamine linker, and 1,2-epoxydodecane as the hydrocarbon tail.

14. The method of claim 12, wherein the ionizable lipid is about 25% to about 45% of the total composition, DOPE is about 10% to about 25% of the total composition, cholesterol is about 30% to about 60% of the total composition, and DMPE-PEG is about 0.5% to about 3.5% of the total composition.

15. The method of claim 14, wherein the ionizable lipid is about 35% of the ionizable lipid of claim 1, DOPE is about 10% of the total composition, cholesterol is about 53.5% of the total composition, and DMPE-PEG is about 1.5% of the total composition.

16. The method of claim 12, wherein the agent is selected from the group consisting of a mRNA, siRNA, microRNA, short hairpin RNA (shRNA), DNA, peptide, and protein.

17. The method of claim 12, wherein at least one agent is mRNA.

18. The method of claim 12, wherein the disorder is a placental related disorder.

19. The method of claim 18, wherein the disorder is preeclampsia.

20. A method of delivering an agent to a hypoxic tissue in a subject comprising administering the composition of claim 8 to the subject in need thereof, wherein the agent is delivered to the hypoxic tissue.

21. The method of claim 20, wherein the ionizable lipid comprises 2-nitroimidazole as the head group, 1,3-diaminopropane as the polyamine linker, and 1,2-epoxydodecane as the hydrocarbon tail.

22. The method of claim 20, wherein the ionizable lipid is about 25% to about 45% of the total composition, DOPE is about 10% to about 25% of the total composition, cholesterol is about 30% to about 60% of the total composition, and DMPE-PEG is about 0.5% to about 3.5% of the total composition.

23. The method of claim 22, wherein the ionizable lipid is about 35% of the ionizable lipid of claim 1, DOPE is about 10% of the total composition, cholesterol is about 53.5% of the total composition, and DMPE-PEG is about 1.5% of the total composition.

24. The method of claim 20, wherein the agent is selected from the group consisting of a mRNA, siRNA, microRNA, short hairpin RNA (shRNA), DNA, peptide, and protein.

25. The method of claim 20, wherein at least one agent is mRNA.

26. The method of claim 20, wherein the hypoxic tissue is the placenta.

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